The induction of chromosome aberrations in human purified peripheral blood lymphocytes following in vitro exposure to selenium

Human lymphocyte cultures were treated with increasing concentrations (8.0 X 10(-8) M to 8.0 X 10(-5) M) of sodium selenite and selenomethionine 24 h after stimulation with phytohemagglutinin and were scored for chromosomal aberrations at 48 h. The yield of abnormal metaphases was dependent on the dose and the form of selenium used. At 8.0 X 10(-5) M the proportion of aberrant cells reached 53.5% and 43.0% for selenite and selenomethionine, respectively. The selenium-induced chromosomal aberrations were primarily of the chromatid type and included breaks and fragments. Chromosomal exchanges were less frequent and included triradials and quadriradials. These results confirm that selenium is clastogenic for cultured human lymphocytes.     

Cytogenetic effects of methotrexate on human cells in vivo: comparison between results obtained by chromosome studies on bone-marrow cells and blood lymphocytes and by the micronucleus test

Cytogenetic studies were performed in 22 patients treated with methotrexate (MTX). In some patients, metaphases from both bone-marrow cells and peripheral blood cells were studied. In the bone-marrow preparations an increased number of structural chromosomal aberrations was present, whereas abnormalities were not observed in the peripheral blood cells. An examination of the bone-marrow chromosomes must therefore be included in the study of the possible chromosome-breaking effect of chemical agents. The results obtained with the micronucleus test and chromosome studies were compared in 10 patients treated with MTX. The micronucleus test was more sensitive than the chromosome analysis as regards the clastogenic effect of MTX.     

Chromosome and cytome analysis of the somatic cells of nuclear-chemical production workers with incorporated 239Pu

The analysis of plutonium production factors has been carried out by using two methodical approaches: assessment of chromosomal aberrations level in routine and G-banded metaphases and molecular-cytogenetic investigation of aneugenic/clastogenic damages in cytokinesis-block binuclear lymphocytes by FISH with centromere specific DNA probes. The obtaining data point out for the first time about both aneugenic and clastogenic influences of incorporated 239Pu with activity range from 0.37 to 6.95 kBq. Correlation analysis of chromosome aberrations with cytome abnormalities allowed finding significant connection between number parameters of metaphase and interphase approaches. The results of this study support the suggestion that aberrant chromosomes are involved preferable in aneugenic events. The FISH technique in binucleated cytokinesis-blocked lymphocytes allows extending of detecting spectrum of chromosome damages and glance of aneugenic mechanisms. Correlations between metaphase and interphase-FISH results point out a high sensitivity of FISH cytome assay, which could be used as an independent test for detection both clastogenic and aneugenic environment influences.     

Acetylsalicylic acid exhibits anticlastogenic effects on cultured human lymphocytes exposed to doxorubicin

Acetylsalicylic acid (ASA) is a non-steroidal anti-inflammatory drug (NSAID) with many pharmacological properties, such as anti-inflammatory, antipyretic and analgesic. Many studies have suggested the possible efficiency of ASA and other NSAIDs in preventing cancer. ASA could also have antimutagenic and antioxidant properties. The aim of this study was to investigate the possible clastogenic and anticlastogenic effects of different concentrations of ASA on doxorubicin-induced chromosomal aberrations in human lymphocytes. Human blood samples were obtained from six healthy, non-smoking volunteers; and the chromosomal aberration assay was carried out using conventional techniques. The parameters analyzed were mitotic index, total number of chromosomal aberrations and percentage of aberrant metaphases. The concentrations of ASA (25, 50 or 100 microg/mL) tested in combination with DXR (0.2 microg/mL) were established on the basis of the results of the mitotic index. The treatment with ASA alone was neither cytotoxic nor clastogenic (p>0.01). In lymphocyte cultures treated with different combinations of ASA and DXR, a significant decrease in the total number of chromosome aberrations was observed compared with DXR alone (p<0.01). This protective effect of ASA on DXR-induced chromosomal damage was obtained for all combinations, and it was most evident when ASA was at 25.0 microg/mL. In our experiments, ASA may have acted as an antioxidant and inhibited the chromosomal damage induced by the free radicals generated by DXR. The identification of compounds that could counteract the free radicals produced by doxorubicin could be of possible benefits against the potential harmful effects of anthracyclines. The results of this study show that there is a relevant need for more investigations in order to elucidate the mechanisms underlying the anticlastogenic effect of ASA.     

A comparison of aberration distribution and cell-cycle progression in cells treated with bleomycin with those exposed to X-rays

The extent of cell-cycle delay and the frequency of aberrant metaphases induced by bleomycin (BLM) and X-rays have been compared at doses which produce similar frequencies of chromosome aberrations by the 2 clastogenic agents (BLM, 40 micrograms/ml and X-rays, 2 Gy) in muntjac lymphocytes. The frequency of aberrant metaphases was low in BLM-treated cells; however, the number of aberrations per metaphase was higher than in cells exposed to X-rays. Thus in contrast to their uniform sensitivity to X-rays, the lymphocytes showed differential sensitivity to BLM. This might be due to differences among the cells in their uptake of BLM and/or its action on the nuclear membrane-DNA complex. In spite of the total number of chromosome aberrations being similar to that induced by X-rays, BLM did not induce a significant delay in cell-cycle progression as observed in the case of X-rays. A possible explanation could be that the DNA damages being limited to fewer cells than in the case of X-irradiation, the BLM-treated cultures had more normal cells allowing faster progression and/or unlike X-rays BLM may not be causing other cellular damages in addition to DNA breaks.     

Effects of the antioxidants curcumin and vitamin C on cisplatin-induced clastogenesis in Wistar rat bone marrow cells

The use of dietary antioxidants to prevent antitumor agent-induced chromosomal damage in nontumor cells is currently eliciting considerable interest. Curcumin (CMN) is a dietary antioxidant that has been reported to protect against clastogenesis in in vivo and in vitro assays. This study was undertaken to investigate the modulatory effects of CMN on cisplatin-induced chromosomal aberrations in Wistar rat bone marrow cells and whether there is any potentiation of these effects with the combination between CMN and vitamin C (VC), which has been reported to reduce the clastogenic effect of many antitumor agents in in vivo assays. Animals treated with CMN plus a single dose of cisplatin, at 18, 24 or 72 h following treatment, presented a statistically significant reduction in the total amount of chromosomal damage and in the number of abnormal metaphases. The results also indicate that the combination between antioxidants would not be effective in protecting against cisplatin-induced chromosomal damage in animals sacrificed 24 h after cisplatin treatment. Under the present experimental conditions, CMN could prevent cisplatin-induced clastogenesis by acting as a free radical scavenger.     

Direct analysis of the clastogenic effect of formaldehyde in unstimulated human lymphocytes by means of the premature chromosome condensation technique

The cytogenetic effect of formaldehyde (FA) on unstimulated human lymphocytes was studied by means of conventional chromosome analysis and the premature chromosome condensation (PCC) technique. In first post-treatment metaphases no significantly increased yields of chromosomal changes could be observed. The analysis of PCCs, however, showed high yields of chromosome fragments. Bleomycin (BLM) used as positive control was also highly clastogenic in PCCs and resulted in significantly increased yields of chromosome-type aberrations. As recently argued, a premitotic selection against heavily damaged cells could be an explanation for the discrepancy between the chromosome findings in metaphase and PCC analysis after FA treatment. In addition, a differential effectiveness may exist in unstimulated lymphocytes to convert multiple fragmentation into chromatid- or chromosome-type aberrations through S-phase-dependent or S-phase-independent mechanisms.     

Chromosomal changes induced by chrysotile fibres or benzo-3,4-pyrene in rat pleural mesothelial cells

The induction of chromosomal aberrations in rat pleural mesothelial cells (RPMC) following in vitro treatment with chrysotile fibres has been demonstrated. The production of chromosomal aberrations was also observed after treatment of the cells with benzo-3,4-pyrene (BP). The yield of abnormal metaphases was dose-dependent and reached 58% at a BP dose of 2 micrograms/ml. Chrysotile fibres at 7 micrograms/ml induced 21% abnormal metaphases and the frequency decreased with further increases in fibre concentration. Their decline is possibly related to a lethal effect. Chrysotile-induced chromosomal aberrations were primarily of the chromatid type and included breaks and fragments. BP induced chromosome exchanges which were not seen following chrysotile treatment. Minutes and double minutes were detected in BP-treated RPMC and occasionally found after chrysotile application. These results confirm that chrysotile fibres are clastogenic for some cultured cells and demonstrate that the fibres induce chromosome damage in target RPMC.     

Influence of treatment to sacrifice time and the presence of BrdUrd on chemically-induced aberration rates in mouse marrow cells

4 chemicals, with various modes of clastogenic action were used to evaluate induced chromosomal aberrations in mouse bone marrow at different times after intraperitoneal injection. Aberration frequencies induced by mitomycin C, cyclophosphamide and dimethylbenz[a]anthracene increased with increasing time between treatment and sampling until those time points (approximately 18 h) when significant proportions of second-division metaphases were among the cells being scored; this increase was not obvious following treatment with 4-nitroquinoline 1-oxide. When BrdUrd tablets were implanted prior to treatment and scoring was restricted to first-division metaphases, aberration rates continued to increase for as long as 24 h post-treatment. The presence of BrdUrd did not affect significantly the rate of aberration induction by the chemicals. Our data indicate that the sensitivity of the in vivo mouse marrow assay for clastogenic chemicals can be greatly increased by utilizing BrdUrd to insure the scoring of only first-division metaphases at post-treatment times of approx. 18 h.     

Clastogenicity of carbazole in mouse bone marrow cells in vivo

Clastogenicity of carbazole was evaluated by employing mouse in vivo chromosomal aberration (CA) test. Carbazole administered intraperitoneally (i.p.) at the rate of 25, 50, 100, 150 and 200 mg/kg b.w. to Swiss albino mice in vivo resulted in mitotic depression and induction of chromosomal aberrations. Dose related decrease in mitotic index (MI) and increase in the frequencies of chromosomal aberrations per cell (CAs/cell) and percent abnormal cells were recorded in bone marrow cells. However, statistically significant reduction in MI and increase in CAs/cell and percent abnormal cells were found only for the two higher doses. The results obtained indicate that carbazole or its metabolite, if any, is moderately clastogenic in the bone marrow cells of Swiss albino mice.     

The effects of increasing dosages of X-radiation on the chromosomes of the central mudminnow, Umbra limi (Kirtland) (Salmoniformes: Umbridae)

Fish subjected to 350 R, 660 R and 990 R of X-radiation showed chromosomal aberrations such as chromatid breaks and gaps, and chromatid exchanges between several chromosomes. The frequency of aberrations/metaphase increased with radiation dosage. Likewise, the percentage of aberrant cells increased with increased irradiation. The countable metaphases fish was lower for higher doses of radiation. At lower doses single chromatid breaks accounted for most of the aberrations whereas complex aberrations involving the breakage and exchange of fragments between several chromosomes were more frequent in fish subjected to 990 R. Gill tissue yielded three times as many countable metaphases as did spleen tissue.     

Induction of chromosomal aberrations, sister-chromatid exchanges and cell-cycle delay in cow peripheral blood lymphocytes treatment with two N-nitroso compounds in vitro

We investigated the effect of NDMA and DNSGU on the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs), as well as the influence of the former compound on cell-cycle kinetics in cultured cow peripheral lymphocytes. A clastogenic effect was observed in treated cell cultures at 6 or 12 × 10⁻⁵ M concentrations of NDMA and DNSGU, respectively, but no increase of chromosomal breaks was seen at the lowest dose. NDMA at 6 × 10⁻⁴ M was toxic to cow lymphocytes. NDMA and DNSGU induced statistical increases of SCEs at the test doses (6 or 12 × 10⁻⁶ and 6 or 12 × 10⁻⁵ M, respectively). In addition, treatment with NDMA at a dose of 6 × 10⁻⁵ M revealed significant heterogeneity of the first, second and third metaphases between treated and untreated groups. A reduction of the proliferation index and proliferation delay per cycle was shown too.     

Mechanism of the chromosome damaging effect of ftorafur]

Chromosome damage induced by three antineoplastic drugs -- ftuorafur (Ft), 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (FUdR) hase been studied in Djungarian hamsters cell line after 24 hours exposition with these agents before the fixation. Ft at a dose of 10 micron/ml induced aberrations in 56.7% of metaphases. 60.0% of aberrant metaphases were obtained in experiments with 0.1 micron/ml of 5 FU. FUdr at the same dose induced 24.0% of aberrant metaphases. The high frequency of chromatid breaks and gaps was typical for the mutagenic action of these fluorinated pyrimidines. The addition of Ft or 5-FU to the cell cultures 1--12 hours before fixation did not produce any significant chrosome damage, while further exposition with the drugs for 15--24 hours caused breaks in more than 50% of metaphases. Thymidine at a concentration of 1.0 micron/ml suppressed the chromosome breaking effect of Ft and 5-FU. The results obtained are in accordance with the idea that fluorodeoxyruidinemonophosphate is the ultimate mutagen for both Ft and 5-FU and that the aberrations observed are due to the lack of thymidine nucelotides caused by the former agents while DNA replication.     

Chromosome aberrations induced by 9-aminoacridine derivatives

The induction of chromosomal aberrations by 5 derivatives of nitro-9-aminoacridine in V79 Chinese hamster cells was observed. The clastogenic activity of the compounds tested depended on the position of the NO2 group in the acridine ring. The strongest clastogens were derivatives with NO2 in position 1. The remaining derivatives placed in decreasing order of clastogenic activity were: 3-nitro, 4-nitro and 2-nitro. In addition, 2-nitro and 3-nitro derivatives produced hyperdiploid/polyploid metaphases.     

Decreased clastogenicity of dinitropyrenes in Chinese hamster lung (CHL) subclone cells with low NADPH-cytochrome P-450 reductase activity

Clastogenic potentials of 1,3-, 1,6- and 1,8-dinitropyrenes (DNPs) were compared between Chinese hamster lung (CHL) cells and its subclone MM1 cells, which were recently isolated as menadione-resistant cells after treatment with MNNG. NADPH-cytochrome P-450 reductase activity of the MM1 cells decreased to 50% of that in the parental CHL cells. All 3 DNPs induced chromosomal aberrations without exogenous metabolic activation systems in the CHL cells. 1,6- and 1,8-DNP showed equivalent clastogenic potency: the maximum frequency of cells with chromosomal aberrations was about 50% for both chemicals. The clastogenic potential of 1,3-DNP was lower than that of 1,6- and 1,8-DNP: the maximum frequency of aberrant cells was 10%. In the MM1 cells, in contrast, the frequencies of aberrant cells decreased to about 30% of those observed for the parental CHL cells after treatment with 1,6- and 1,8-DNP, and to the same level as that of the concurrent control after treatment with 1,3-DNP. These results suggest a possibility that the reduced clastogenic effect of 3 DNPs in MM1 cells may correlate with the reduced activity of NADPH-cytochrome P-450 reductase which is thought to contribute to the metabolic conversion of these DNPs to their clastogenic forms in the CHL cells.     

The analysis of chromosome lesions in lymphocytes of Hodgkin's lymphoma patients after chemotherapy and radiation therapy

The analysis of chromosome lesions in peripheral blood lymphocytes of Hodgkin's lymphoma (HL) patients after chemotherapy and chemotherapy with the subsequent course of radiation therapy is carried out. Is shown, that the mean aberration frequency was significantly higher in HL patients after chemotherapy (7.20 +/- 0.58 per 100 metaphases) than in non-treated HL patients (4.80 +/- 0.54, p < 0.01). The subsequent carrying out of radiation therapy enlarges number of chromosome aberrations on 100 metaphases up to 46.7 +/- 10.7 (p < 0.05), of which chromosome-type aberrations (43.2 +/- 10.3 on 100 metaphases) averaged 92.5%. In lymphocytes of 37 out of 43 HL antitumoral treatment patients, we found, in addition to ordinary aberrant cells, a large number of multiaberrant (MA-cells) cells, i.e. metaphases carrying multiple (at least four) chromosome-type exchange aberrations. In 30 non-treated HL patients only one MA-cell was found. From 171 MA-cells which were in 43 HL patients after antitumoral treatment, 114 MA-cells were found at inspection of 9766 diploid metaphases, and the remaining 57 MA-cells were found at inspection of 196 polyploid metaphases. The carrying out after chemotherapy of radiation therapy enlarges in lymphocytes frequency of appearance of MA-cells. The analysis of MA-cells in diploid and polyploid metaphases shown, that the MA-cells could be formed both in vivo, and in vitro in absence of influence of clastogenic factors, and could survive at least two rounds of in vitro replication.     

Clastogenic action of ellipticine over the cell cycle of human lymphocytes and influence of posttreatments with caffeine and ara-C at G2

The clastogenic potential of the intercalating compound ellipticine, an antitumor alkaloid, has been demonstrated in mammalian cells. To characterize the mechanism of action of this drug over the cell cycle, human lymphocyte cultures from 2 healthy donors were treated with 3 micrograms/ml ellipticine in 30-min pulses during different phases of the cell cycle and analyzed for chromosomal aberrations and sister-chromatid exchanges. The G2 phase was most sensitive in terms of induction of aberrations, followed by S and G1. Chromatid-type aberrations were the most common type of chromosomal damage. Induction of SCEs was significantly high only after treatment at G1, when the frequencies of SCEs doubled. The post-treatment effect of lymphocytes with inhibitors of DNA repair, 10(-3) M caffeine and 5 x 10(-6) M 1-beta-D-arabinofuranosylcytosine, was also tested by adding 3 micrograms/ml ellipticine at G2 in 30-min pulses and immediately followed by caffeine and/or ara-C during the last 3 h before harvesting. Three experiments performed on blood from 3 donors showed a moderate potentiation effect on the frequency of chromatid-type aberrations (about 2-3 times) by both inhibitors. Likewise, a 3-fold increase was observed in the frequencies of chromosomal aberrations when caffeine and ara-C were combined. The present data demonstrate that posttreatment with caffeine and ara-C at G2 can modify the response of human lymphocytes treated with ellipticine by increasing the clastogenic action of this compound or by changing the cell-cycle progression.     

Chromosome-damaging action of isoniazid and thiacetazone on human lymphocyte cultures in vivo

Summary Antitubercular drugs in general are given in various combinations, one being isoniazid and thiacetazone. In the present study, was evaluated the in vivo chromosome-damaging effects of a combination of these two drugs in 72 h lymphocyte cultures.Chromosome aberrations were significantly increased in the patients treated with INH and thiacetazone as compared with two types of controls: (1) tuberculosis patients before starting the drug treatment and (2) individuals from the general population. The most frequently observed aberrations were chromatid breaks and gaps.It has been shown that individually, isoniazid may not be clastogenic on human chromosomes in therapeutic doses. The effects of thiacetazone on human chromosomes are not known. Consequently, the enhancement in chromosomal aberrations in the drug-exposed patients may be due to a synergistic effect of isoniazid and thiacetazone or to the clastogenic effects of thiacetazone alone.     

Severe chromosomal damage in mammalian cells induced by pulse labeling with small amounts of [3H]thymidine

Pulse labeling of cultured mammalian cells with tritiated thymidine ([3H]TdR; different specific activities, 2-47 Ci/mmole) for 30 min at low concentrations of radioactivity (0.2-2 microCi/ml) causes an unusually high degree of chromosomal aberrations which becomes fully evident in the cohort of cells analyzed 8 h later. The effect depends on the total amount of radioactivity. In HeLa cells up to 93% aberrant metaphases with up to 3.6 aberrations per metaphase were observed; in CHO cells the corresponding figures are 62% aberrant metaphases with 1 aberration per metaphase. The results may be valuable (i) for users of the pulse-labeling technique for analysis of cell-cycle kinetics, as this shows the technique as such may delay the labeled cohort of cells and therefore interferes with the matter under study and (ii) for risk assessment.     

Induction of chromosomal aberrations by camptothecin in root-tip cells of Vicia faba.

When root-tip cells of Vicia faba were exposed during early and middle interphase to camptothecin (Cpt), the aberrations obtained were exclusively of the chromatid type and tended to be localized in late replicating heterochromatic regions of the chromosomes. In these respects the clastogenic effect of Cpt resembles that of agents that act by an S-phase-dependent mechanism. In contrast to typical S-phase-dependent agents, Cpt produced lesions capable of giving rise to aberrations only in S-phase cells that were synthesizing DNA at the time of treatment. The dependence on ongoing DNA synthesis was suggested in autoradiographic experiments and by the fact that the clastogenic effect of Cpt was strongly suppressed by hydroxyurea, an inhibitor of DNA synthesis. After Cpt treatments, there were many more cells with 3-12 aberrations and far fewer cells with 0, 1 or 2 aberrations than expected on the basis of a Poisson distribution. Cpt further differed from typical S-phase-dependent agents by being capable of inducing lesions in the G2 phase that give rise to chromosomal aberrations in the first mitosis after treatment. This effect was obtained at Cpt concentrations around 10 microM, whereas only 0.03 microM Cpt was required to produce chromatid aberrations in the S phase. Results of G2-phase experiments with Cpt and 5-fluorodeoxyuridine, an inhibitor of DNA synthesis, suggest that DNA synthesis is required for the clastogenic effect of Cpt not only during the S phase, but also during the G2 phase, although the DNA syntheses involved are both quantitatively and qualitatively different.